The present invention relates especially to very highly specific surfaces which can be used in biology, as well as to their applications and to processes for preparing them.
The very high specificity and the very high selectivity of certain biological reactions, especially antigen/antibody reactions, DNA or RNA hybridization reactions, interprotein or avidin/streptavidin/biotin type reactions, as well as reactions of ligands and their receptors, have been known for a long time.
It is now known how to take advantage of these specificities, especially in order to detect the presence or the absence of one of the elements of the reaction pair in a sample or alternatively for separating one of the elements of the pair from a more complex medium.
However, when it is desired to detect the presence of a molecule at a very low concentration in a very complex medium, currently known processes sometimes give very unpredictable results given especially the problem of background noise which occurs during the separation and/or detection stages.
Consequently, what will be called hereinafter "molecular fishing", that is to say the possibility of being able to detect each of the searched-for molecules when they are at very low concentrations, has so far not been possible.
By way of example, the analysis of a DNA sample requires the use of a so-called "hybridization" probe corresponding to the sequence complementary to the desired sequence. Under these conditions, the problem posed is to isolate the hybrid from the medium and to detect, with a good signal/noise (S/N) ratio, the possibly reduced number of positive reactions.
Consequently, an intermediate stage intended to amplify the sought sequence is now used in most cases, for example using the PCR method or amplification methods which lead to the same results; under these conditions, the concentration of the sequence to be determined is increased in the sample and its detection is obviously much easier.
However, the amplification stage is sensitive to contaminants and leads to errors which are specific to it.
It would therefore be preferable, as far as possible, to be able to detect the presence of the nucleic acid sequence without an amplification phase.
It has been proposed to use, in order to detect the specific hybridization reaction, an intermediate stage of anchoring the hybridization product on a solid surface having certain specificities. For example, it is possible to use certain pretreated surfaces which make it possible to attach certain proteins or DNA, whether it has been modified or not.
Such surfaces are commercially available (Covalink, Costar, Estapor, Bangs, Dynal for example) in the form of beads or wells having at their surface COOH, NH.sub.2 or OH groups for example.
It has also been proposed, in order to obtain such groups, to use an intermediate stage having a vinyl group which is then oxidized so as to have COOH or OH groups (U.S. Pat. No. 4,539,061 and EP 435 785).
It is then possible to functionalize the DNA with a reactive group, for example an amine, and to carry out a reaction with these surfaces. These methods require, however, a specific functionalization of the DNA to be attached.
A technique has also been described permitting the anchoring without prior treatment of the DNA. This process consists in reacting the free phosphate of the 5' end of the molecule with a secondary amine (NH Covalink surface).
It is also possible to attach the DNA to a group or a protein P.sub.0 in order to cause it to react with a surface coated with a group or a protein P.sub.1, which is capable of reacting specifically with P.sub.0. The P.sub.0 /P.sub.1 pair may be a pair of the following type: biotin/streptavidin or digoxigenin/antibody directed against digoxigenin (anti-DIG) for example.
Such surfaces are, however, in most cases insufficiently specific (V. Lund et al., Nucl. Acids Res., 16, 1861 (1988)). Thus, the presence of unwanted, even weak, interactions of the nonspecific adsorption type leads to efficient adsorptions for long molecules capable of forming, with the solid, a large number of points of weak interaction. These surfaces lead to potential applications which lack sensitivity and/or with a high level of background noise in the case of a small number of molecules to be fished out. Furthermore, some of these surfaces have a high level of unwanted fluorescence which is potentially disruptive during the detection phase.
As regards the detection itself, in particular for the detection of DNA, French Patent 78 10975 describes a process coupling the probe with an enzyme which allows revealing by means of a chromogenic substrate. It is, in addition, possible to quantify the reaction by a calorimetric measurement.
Such a technique is, however, not directly adapted to the detection of traces; consequently, here too, it should be preceded in most cases by an amplification stage for the desired quantity of nucleic acid, for example by the PCR method.
This so-called process of detection "by a cold probe" was developed in order to avoid the use of radioactive markers which yield results close in terms of sensitivity but which obviously present handling problems, given the presence of radioactive products and problems of long revealing times if a high sensitivity is desired.
For certain specific applications, especially methods derived from ex vivo imaging, there has been proposed a direct method for observing the reaction by coupling the product of the hybridization to microbeads, especially PMMA, polymethylmethacrylate, suitably treated chemically at their surface. The method is based on the direct identification, under a scanning electron microscope, of the presence of these microbeads with a typical diameter of 60 nm, and is furthermore based on known but insufficiently specific techniques for anchoring on solids, as described above.
The above techniques are obviously not limited to the detection of nucleic acids. In the same spirit, the detection of antibodies has been proposed. They are ELISA type tests which will not be described here and which, to summarize, make it possible to couple the presence of an antibody to an associated anchoring of a molecule of antigen on a solid. Again, the problems of specificity and unwanted reactions exist. The detection phase can then be based on a coupling to a chromogenic reaction having its own problems of sensitivity.
In summary, the prior art methods or combinations thereof have a number of disadvantages, in particular:
either of being potentially dangerous because of the use of radioactive products, PA1 or of requiring revealing times which are too long, PA1 or of being disrupted by specific problems at the level of the amplification phase, PA1 or of requiring solid surfaces which are not sufficiently specific PA1 or of being too weakly sensitive, PA1 or, finally, of requiring, in addition to the phase for attachment to a solid, the use of an electron microscope which is obviously not very convenient.
Finally, in most cases, the known processes do not make it possible to recognize, on a given molecule, the specific position of the desired unit. Now, this type of recognition is important when it is desired to perform mapping, in particular within the framework of genome mapping, it is desired to recognize, in a first instance, the approximate spatial position relative to one end of the molecule of a given gene on a DNA or an RNA.